There are a number of modifications that proteins can undergo following translation. Some of the many post-translational modifications result in proteolytic processing or the covalent linkage of an important functional group to the protein. An interesting post-translational modification is the head-to-tail cyclization of a protein or peptide to form a continuous peptide backbone. Many of the naturally occurring circular peptides posses anti-bacterial activity, such as the AS-48 peptide (Samyn, et al., FEBS Lett., 352(1) 87-90 (1994)). Also, these antibacterial peptides have been found in organisms as divergent as bacteria and primates (Samyn, et al., FEBS Lett., 352(1) 87-90 (1994); Tang, et al., Science, 286(5439) 498-502 (1999)). One possibility for forming a cyclic protein species may be that the peptide or protein is more conformationally stable once its N- and C-termini have been constrained.
In addition to the naturally occurring cyclic peptides a number of synthetic techniques have been developed to generate synthetic circular peptides (Tam and Lu, Protein Sci., 7(7) 1583-1592 (1998); Romanovskis and Spatola, J. Pept. Res., 52(5) 356-374 (1998); Camarero and Muir, J. Amer. Chem. Soc., 121 5597-5598 (1999); Valero, et al., J. Pept. Res., 53(1) 56-67 (1999)). However, due to the limitations of total chemical synthesis it is difficult to generate synthetic cyclic peptides larger than 100 amino acids. This was circumvented using intein based technologies that allowed ribosomally synthesized proteins to cyclize in a head-to-tail fashion in vitro (Camarero and Muir, J. Amer. Chem. Soc., 121 5597-5598 (1999); Evans, et al., J. Biol. Chem., 274 18359-18363 (1999); Iwai and Pluckthun, FEBS Lett., 459 166-172 (1999)). However, these procedures did not allow the cyclization of a protein or peptide in vivo for study in a living organism.
The in vitro cyclization of ribosomally synthesized proteins utilize the activity of protein splicing elements (termed inteins Perler, et al., Nucleic Acids Res., 22 1125-1127 (1994)). Inteins, catalyze their own excision from a primary translation product with the concomitant ligation of the flanking protein sequences (reviewed in Paulus, Chem. Soc. Rev., 27:375-386 (1998), Perler, Cell 92(1)1-4 (1998) and Shao and Kent, Chem. Biol. 4(3):187-194 (1997)). Inteins catalyze three highly coordinated reactions at the N- and C-terminal splice junctions (Xu and Perler, EMBO J. 15(19):5146-5153 (1996) and Chong, et al., J. Biol. Chem., 271:22159-22168 (1996)): 1) an acyl rearrangement at the N-terminal cysteine or serine; 2) a transesterification reaction between the two termini to form a branched ester or thioester intermediate; and 3) peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein. Inteins have been engineered to be versatile tools in protein purification (Chong, et al., Gene, 192(2) 271-281 (1997), Chong, et al., Nucleic Acids Res. 26(22):5109-5115 (1998), Evans, et al., Protein Sci., 7:2256-2264 (1998), Mathys, et al., Gene 231:1-13 (1999), Evans, et al., J. Biol. Chem., 274:3923-3926 (1999), Southworth, et al., Biotechniques, 27:110-120 (1999) and Wood, et al., Nature Biotechnology, 17(9):889-892 (1999)), protein ligation (Evans, et al., Protein Sci., 7:2256-2264 (1998), Mathys, et al., Gene 231:1-13 (1999), Evans, et al., J. Biol. Chem., 274:3923-3926 (1999), Southworth, et al., Biotechniques, 27:110-120 (1999), Cotton, et al., J. Ant. Chem. Soc. 121:1100-1101 (1999), Muir, et al., Proc. Natl. Acad. Sci. USA. 95:6705-6710 (1998), Severinov and Muir, J. Biol. Chem. 273:16205-16209 (1998), and Xu, et al., Proc. Natl. Acad. Sci. USA 96(2):388-393 (1999)) as well as in the aforementioned formation of cyclic proteins and peptides (Evans, et al., J. Biol. Chem. 274:18359-18363 (1999), Iwai and Pluckthun, FEBS Lett 459:166-172 (1999) and Camarero and Muir, J. Amer. Chem. Soc., 121:5597-5598 (1999)). Limitations of these intein technologies include the necessity of generating an N-terminal cysteine and/or C-terminal thioester intermediate in vitro for ligation or cyclization, the need to perform extra purification steps to separate unligated reactants from the ligation products and the requirement of a denaturant to permit in vitro trans-splicing reactions (Yamazaki, et al., J. Am. Chem. Soc. 120:5591-5592 (1998), Mills. et al., Proc. Natl. Acad. Sci. USA, 95(7):3543-3548 (1998), and Southworth, et al., EMBO J., 17(4):918-926 (1998)).
In addition to the cis-splicing inteins and those engineered to trans-splice (Yamazaki, et al., J. Am. Chem. Soc. 120:5591-5592 (1998), Wu, et al., Biochim. Biophys Acta, 1387:422-432 (1998), Mills, et al., Proc. Natl. Acad. Sci. USA, 95(7):3543-3548 (1998), Otomo, et al., J. Biomol. NMR, 14(2):105-114, Otomo, et al. Biochemistry, 39(49):16040-16044, and Southworth, et al., EMBO J., 17(4):918-926 (1998)), a naturally-occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (Wu, et al., Proc. Natl. Acad. Sci. USA, 95(16):9226-9231 (1998)). The N-terminal half of DnaE, followed by a 123-amino acid intein sequence, and the C-terminal half, preceded by a 36-amino acid intein sequence, are encoded by two open reading frames located more than 745 kilobases apart in the genome. When co-expressed in E. coli, the two DnaE-intein fragments exhibited protein trans-splicing (Wu, et al., Proc. Natl. Acad. Sci. USA, 95(16):9226-9231 (1998)).
Accordingly, it would be desirable to utilize intein technology in developing methods for producing circular or multimeric protein species in vivo or in vitro. Such methods would permit the formation of cyclic polypeptides in new hosts, facilitate the separation of products from reactants when ligating proteins for isotopic labeling, and allow the generation of cyclic polypeptides that are sensitive to reducing agents.